The overall aim of this research is to identify and characterize gene regulation events that propel rate-limiting steps during tumor promotion and tumor progression. The AP-1 transcription factor is a heterodimer of Jun and Fos family proteins that binds to a specific sequence on the transcriptional promoter of certain genes and drives their transcription. Our 1989 observation (Bernstein and Colburn, Science, 1989) that transformation sensitive (P+) but not transformation resistant (P-) mouse JB6 cells responded to tumor promoters by activating AP-1 dependent transcription, suggested that AP-1 activation might be required for progression from preneoplastic to neoplastic (tumor) phenotype. A requirement for AP-1 dependent gene transcription was established by the observation that expression of dominant negative jun (TAM67) blocked both AP-1 activation and transformation response in mouse JB6 cells (Dong et al PNAS, 1994). Furthermore transgenic mice expressing the K14-TAM67 transgene show 90% inhibition of AP-1 activation and 90% protection against promotion of skin carcinogenesis, i.e., prevention of premalignant papilloma formation (Young et al., PNAS, 1999). This inhibition of tumorigenesis occurred without evidence of toxicity or inhibition of the hyperplasia response to tumor promoter. Keratinocyte-specific expression of Dominant Negative Jun in transgenic mice inhibits induced AP-1 and tumorigenesis without inhibiting growth, differentiation or hyperplasia in multiple mouse models relevant to human carcinogenesis. Among these are mice whose skin tumor promotion response is elevated by expression of Human Papilloma Virus E7 (Young et al Molec Carc 2002), mice promoted by phosphatase inhibitor okadaic acid (Thompson et al Cancer Res 2002), and mice induced to form squamous carinomas by repeated exposure to UVB (Cooper et al Molec Cancer Res 2003). For further studies, tetracycline regulated expression of TAM 67 is being directed to skin and cervix in our laboratory, and to mammary and lung epithelia in the laboratories of collaborators Powel Brown and Jay Tischlaar. When the transgene K14-TAM67 or a tetracycline regulated TAM67 construct was expressed in the more progressed human cell lines that are tumorigenic or anchorage independent and show elevated AP-1 and NFkB activities, tumor cell phenotype was suppressed (Li et al., Oncogene, 1998 and Li et al., Molec Carcinog 2000). The transcription factor NFkappa B is coordinately regulated with AP-1, suggesting the possible importance of both factors in transformation (Li et, Cancer Res 1997). Recent observations have identified NFkB non-responsiveness as an explanation for transformation non-responsiveness in the JB6 model (Hsu et al, Cancer Res 2001). P- cells owe their transformation nonresponsiveness to an inability to activate NFkappa B p65 protein. Thus the observation that targeting AP-1 and NFkB elevation prevents tumor promotion and progression has been extended from the mouse JB6 model to mouse and human keratinocyte progression models, and to transgenic mouse models. New understanding of critical molecular interactions is emerging. The mouse JB6 cells, the human keratinocytes and the transgenic mice expressing AP-1/ NFkB inhibitor TAM 67 present valuable opportunities to identify AP-1 or NFkB target genes whose expression is critical to neoplastic transformation. Expression microarray analysis has revealed a limited number of potential TAM target genes that are subjects of current research. Such target genes may be promising new molecular targets for cancer prevention (Young et al Trends in Molec Medicine 2003). Recent studies have excluded iNOS (Dhar et al Mol Cancer Ther 2003)and other studies have established the importance of chromatin architectural protein HMGA1(Dhar et al Oncogene in press) as a functionally significant TAM67 target. The transformation resistance of one of the JB6 P- variants is attributable to a shortage of the MAP kinases Erks 1 and 2 which are also limiting for AP-1 transactivation (Huang, et al, PNAS, 1998). Expression of dominant negative Erk 2 in AP-1 responsive cells has demonstrated the requirement of Erk activity for AP-1 activation and transformation responses (Watts et al 1998). Current research directed to identifying Erk dependent molecular events required for activating AP-1 has identified the activation of Fos family protein Fra-1 as a pivotal event (Young et al, Molec Cell Biol 2002). JB6 variants deficient in either Erk or Fra-1 proteins are AP-1 and transformation nonresponsive. Responsiveness is restored by expression of Erk or Fra-1 respectively. A subset of AP-1 dependent gene promoters is expected to require Fra-1 for transcriptional activation and these may serve as particularly effective targets for cancer prevention.